Ingrid 't Hart

48 Chapter 2 2 to give the �tle compound as a white amorphous solid (131 mg, 70 %, over three steps) Addi�onal purifica�on by HPLC with a semi-prepara�ve HILIC column (XBridge® Amide 5 µm, 4.6 mm x 250 mm column, Waters) under isocra�c condi�ons (74% B) with UV detec�on (210 nm) affords analy�cally pure glycan. 1 H NMR (400 MHz, D 2 O) δ 5.21 (0.5H, d, J = 3.7 Hz, H-1α, Glc-I), 4.90 (1H, d, J = 3.8 Hz, H-1, Gal-III), 4.70 – 4.61 (1.5H, m, H-1, GalNAc-IV; H-1β Glc-I), 4.50 (1H, d, J = 7.7 Hz, H-1, Gal-II), 4.45 (1H, d, J = 7.7 Hz, H-1, Gal-V), 4.38 (1H, t, J = 6.3 Hz, H-5, Gal-III), 4.24 (1H, d, J = 2.3 Hz, H-4, Gal-III), 4.17 (1H, d, J = 2.9 Hz, H-4, GalNAc-IV), 4.10 – 4.01 (2H, m, H-2, GalNAc-IV; H-4, Gal-II), 3.99 – 3.54 (23H, m, H-2, Gal-III; H-2, Gal-II), 3.50 (1H, dd, J = 9.8, 7.8 Hz, H-2, Gal-V), 3.27 (0.5H, t, J = 8.4 Hz, H-2, Glc-Iβ), 2.02 (3H, s, NHAc). 13 C NMR (101 MHz, D 2 O) δ 175.0 ( C =O NHAc), 104.7 (C-1, Gal-V), 103.2 (C-1, Gal-II), 102.9 (C-1, GalNAc-IV), 100.3 (C-1, Gal-III), 95.6 (C- 1β, Glc-I), 92.1 (C-1α, Glc-I), 79.5, 78.6, 78.6, 77.1, 75.4, 74.8, 74.5, 74.4, 73.8 (C-2, Glc-I), 72.4, 72.0, 71.4, 71.1 (C-2, Gal-III), 70.81 (C-2, Gal-II), 70.51 (C-2, Gal-V), 70.2, 70.1, 68.9, 68.5, 67.9, 67.5, 60.9, 60.9, 60.3, 60.3, 60.3, 51.4 (C-2, GalNAc-IV), 22.2 (CH 3 , NHAc). ESI HRMS (m/z): [M + Na] + calcd for C 32 H 55 NO 26 , 892.2910; found 892.2912. 5-aminopentyl β-D-Galactopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D- galactopyranosyl-(1 → 3)-α-D-galactopyranosyl-(1 → 4)-β-D-galactopyranosyl-(1 → 4)- β-D-glucopyranoside (4b). Pentasaccharide 16c was deprotected in a total of 5 steps, all steps were the same as for Gb5-OMP, without the CAN reac�on. (4.57 mg, 19 %, over 5 steps). 1 H NMR (400 MHz, D 2 O) δ 4.89 (1H, d, J = 3.5 Hz, H-1, Gal-III), 4.67 (1H, d, J = 8.5 Hz, H-1, GalNAc-IV), 4.54 – 4.40 (3H, m, H-1, Gal-II; H-1, Glc-I; H-1, Gal-V), 4.37 (1H, t, J = 6.3 Hz, H-5, Gal-III), 4.23 (1H, s, H-4, Gal-III) 4.16 (1H, d, J = 2.4 Hz, H-4, GalNAc-IV), 4.10 – 3.46 (28H, m), 3.28 (1H, t, J = 8.2 Hz, H-2, Glc-I), 3.03 – 2.94 (2H, m, CH 2 , pentyl), 2.01 (3H, s, NHAc), 1.76 – 1.58 (4H, m, 2x CH 2 , pentyl), 1.50 – 1.38 (2H, m, CH 2 pentyl). 13 C NMR (101 MHz, D 2 O) δ 175.0 (C=O, NHAc), 104.7 (C-1, Gal-V), 103.2 (C-1, Gal-II), 102.8 (C-1, GalNAc-IV), 101.9 (C-1, Glc-I), 100.3 (C-1, Gal-III), 79.5, 78.7, 78.6, 77.1, 75.3, 74.9, 74.7, 74.5, 74.4, 72.8 (C-2, Glc-I), 72.3, 72.0, 70.8 (C-2, Gal-II), 70.5 (C-2, Gal-V), 70.2, 70.0, 68.8 (C-2, Gal-III), 68.5, 67.9, 67.5, 60.9, 60.8, 60.2, 60.2, 59.9, 51.4 (C-2, GalNAc-IV), 39.2 (CH 2 ), 28.1 (CH 2 ), 26.3 (CH 2 ), 22.2 (CH 3 , NHAc), 22.0 (CH 2 ). ESI HRMS (m/z): [M + H] + calcd for C 37 H 66 N 2 O 26 , 955.3977; found 955.3979. Enzyma�c synthesis Human glycosyltransferase expression The cataly�c domains of human glycosyltransferases (see Table S2 below) were expressed as soluble, secreted fusion proteins by transient transfec�on of HEK293 suspension cultures. 14, 39 The coding regions were amplified from Mammalian Gene Collec�on clones using primers that appended a tobacco etch virus (TEV) protease cleavage site 14, 40 to the NH 2 -terminal end of the coding region and a�L1 and a�L2 Gateway adaptor sites to the 5ʹ and 3ʹ terminal ends of the amplimer products. The amplimers were O HO OH O AcHN O HO OH HO OH O HO OH O HO O O OH HO OH O O OH HO OH O NH 2